Project Summary/Abstract The mTORC1 kinase is a master regulator of cellular growth and metabolism. mTORC1 activity is controlled by upstream environmental cues, including growth factors and nutrient availability; not surprisingly, dysregulation of the mTORC1 pathway has been implicated in a diverse set of diseases, including cancers, diabetes, obesity, neurodegeneration, and the aging process. In order to guide new therapeutic approaches toward these diseases, we must understand how mTORC1 is regulated by specific upstream nutritional inputs on a molecular level. By demonstrating that mTORC1 activity is inhibited by acute serine deprivation, we have identified a new input into the mTORC1 pathway. This result is especially striking because deprivation of serine, a non- essential amino acid, can induce metabolic rewiring in cancer cells and disrupt one-carbon metabolism; moreover, exogenous serine is conditionally essential in many tumors. Our preliminary data show that mTORC1 detects the presence of serine, or a serine-derived metabolite, through the nutrient-sensing pathway upstream of the Rag-GTPases; however, we do not know the identity of the serine sensor, nor do we know how it regulates mTORC1 signaling. To elucidate the mechanism of serine sensing by the mTORC1 pathway, we propose the following specific aims: 1) Establish the pathway upstream of the Rag-GTPases which signals serine sufficiency to mTORC1. 2) Determine the metabolite that signals serine sufficiency to mTORC1. 3) Identify the protein that negatively regulates mTORC1 activity when serine levels are insufficient. We will employ a multidisciplinary approach that incorporates metabolite profiling, unbiased proteomics, and functional genomics to determine how serine promotes activation of the mTORC1 pathway. Through these studies, we may be able to establish a link between altered serine metabolism and mTORC1-dependent growth and proliferation in cancer cells. In addition, our work may uncover new serine-dependent metabolic vulnerabilities in cancers and lead to the identification of novel drug targets in the mTORC1 pathway.